Cerebral Creatine Deficiency Affects the Timing of Oligodendrocyte Myelination

Cerebral creatine deficiency syndrome (CCDS) is an inborn error of metabolism characterized by intellectual delays, seizures, and autistic-like behavior. However, the role of endogenously synthesized creatine on CNS development and function remains poorly understood. Here, magnetic resonance spectroscopy of adult mouse brains from both sexes revealed creatine synthesis is dependent on the expression of the enzyme, guanidinoacetate methyltransferase (GAMT). To identify Gamt-expressed cells, and how Gamt affects postnatal CNS development, we generated a mouse line by knocking-in a GFP, which is expressed on excision of Gamt. We found that Gamt is expressed in mature oligodendrocytes during active myelination in the developing postnatal CNS. Homozygous deletion of Gamt resulted in significantly reduced mature oligodendrocytes and delayed myelination in the corpus callosum. Moreover, the absence of endogenous creatine resulted in altered AMPK signaling in the brain, reduced brain creatine kinase expression in cortical neurons, and signs of axonal damage. Experimental demyelination in mice after tamoxifen-induced conditional deletion of Gamt in oligodendrocyte lineage cells resulted in delayed maturation of oligodendrocytes and myelin coverage in lesions. Moreover, creatine and cyclocreatine supplementation can enhance remyelination after demyelination. Our results suggest endogenously synthesized creatine controls the bioenergetic demand required for the timely maturation of oligodendrocytes during postnatal CNS development, and that delayed myelination and altered CNS energetics through the disruption of creatine synthesis might contribute to conditions, such as CCDS. SIGNIFICANCE STATEMENT Cerebral creatine deficiency syndrome is a rare disease of inborn errors in metabolism, which is characterized by intellectual delays, seizures, and autism-like behavior. We found that oligodendrocytes are the main source of endogenously synthesized creatine in the adult CNS, and the loss of endogenous creatine synthesis led to delayed myelination. Our study suggests impaired cerebral creatine synthesis affects the timing of myelination and may impact brain bioenergetics.


Introduction
The creatine-phosphocreatine shuttle plays an essential role in energy metabolism (Wyss et al., 2007). During periods of high energetic demand, creatine kinases catalyze the transfer of the high-energy phosphate group in phosphocreatine to ADP, allowing for the rapid generation/regeneration of ATP, thereby maintaining the energetic supply required for cellular function (Wyss et al., 2007). About half of our daily creatine is derived from diet. The other half is endogenously synthesized by the conversion of glycine and arginine into guanidinoacetate through the enzyme arginine:glycine amidinotransferase (AGAT) and the subsequent transformation of guanidinoacetate into creatine through the enzyme guanidinoacetate methyltransferase (GAMT). Creatine can then be converted to phosphocreatine and used by cells endogenously or transferred to other cells through the creatine transporter, SLC6A8. Humans with mutations in AGAT, GAMT, or SLC6A8 display cerebral creatine deficiency syndromes (CCDSs), which are rare diseases that are characterized by the disruption of the synthesis or transfer of creatine (Wyss and Kaddurah-Daouk, 2000;Braissant et al., 2011). If left untreated, children with CCDS can present with severe intellectual disabilities, seizures, developmental delays, autistic-like behaviors, and movement disorders, suggesting that the brain is particularly vulnerable to creatine deficiency (Giusti et al., 2019).
Previous studies suggest that two main waves of creatine synthesis occur during rodent CNS development: one from mitotic cells of the subventricular zone and the external layer of the cerebellum, and the second from oligodendrocytes starting at 2 weeks old and continuing into adulthood (Tachikawa et al., 2004(Tachikawa et al., , 2018Baker et al., 2021). The profound increase in postnatal Gamt expression coincides with active CNS myelination (Sturrock, 1980;Baker et al., 2021), and suggests that oligodendrocytes may be a significant source of endogenous creatine within the postnatal brain. During active myelination, oligodendrocytes require a tremendous amount of energy for myelination. An estimated 3.24 Â 10 23 ATP molecules are required to synthesize 1 g of myelin (Harris and Attwell, 2012), and disruption of oligodendrocyte energetic metabolism (i.e., from hypoglycemia) causes a significant delay in CNS myelination (Yan and Rivkees, 2006;Rinholm et al., 2011). We have previously found that creatine protects oligodendrocytes from mitochondrial-mediated apoptosis during injury, and promotes remyelination (Chamberlain et al., 2017). However, whether endogenously synthesized creatine is necessary for developmental myelination or remyelination remains unclear. Since myelination is an energetically demanding process, we hypothesized that oligodendrocyte-derived creatine is required for developmental myelination and remyelination.
Here, we show that GAMT is the primary enzyme responsible for endogenously synthesized creatine in the mouse CNS. To track creatine production in the CNS, we developed a novel transgenic floxed mouse line that expresses GFP on the conditional excision of one or both copies of Gamt, to allow the tracking of cells which produce creatine, and for the analysis of Gamt loss of function in the mouse CNS, respectively. We found that oligodendrocytes are the main cells expressing GFP in the developing CNS, and that Gamt deletion resulted in delayed myelination and remyelination. Additionally, Gamt deletion may lead to altered neuronal bioenergetics in the adult brain. Our results indicate that oligodendrocytes are the major producers of creatine in the adult CNS, supporting previous observations (Tachikawa et al., 2018;Baker et al., 2021), and suggest that oligodendrocyte dysfunction through the loss of Gamt expression might contribute to conditions of creatine deficiency, such as CCDS.

Materials and Methods
Mice. All transgenic mice were maintained on a C57BL/6 background, and experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee at Georgetown University. Mice of both sexes were used for each experiment. GAMT -/1 mice for magnetic resonance spectroscopy (MRS) were a kind gift from Dirk Isbrandt (University of Cologne) (Schmidt et al., 2004) and back-crossed on C57BL/6 for four generations before breeding for MRS experiment. Floxed Gamt (Gamt fl/fl ) line was engineered by Cyagen Biosciences where a linearized vector was electroporated into embryonic stem cells (C57BL/6). After confirming clones by Southern blotting, a chimera was produced by blastocyst microinjection. Floxed Gamt line was bred with Tg(CMV-cre)1Cgn (stock #006054) line from The Jackson Laboratory to first generate a heterozygous mutant (Gamt GFP/1 ). Heterozygous mutants were bred together to initially generate the KO mutant (Gamt GFP/GFP ). For breeding experimental animals, female Gamt GFP/1 dams were bred with male Gamt GFP/GFP animals. Germline removal of Gamt allowed for removal of the excised allele without the passage of Cre transgene in our final experimental Gamt GFP/1 and Gamt GFP/GFP animals. Controls were Gamt fl/fl . Tg(Pdgfra-cre/ERT)467Dbe (stock #018280) from The Jackson Laboratory was used to generate inducible removal of Gamt from oligodendrocytes (OL Gamt iKO) with heterozygous Cre transgene. WT C57BL/6J (stock #000664) mice were also obtained from The Jackson Laboratory. Mice were maintained on a 12 h light/dark cycle with food and water ad libitum. All animals were fed a creatine-deficient amino acid diet (Crt def diet; Envigo; TD.01,084) unless otherwise specified (more details below).
MRS. Animals underwent small animal imaging at the Preclinical Imaging Research Laboratory and the Center for Translational Imaging at Georgetown-Lombardi University Medical Center in a Bruker 7T/ 20 Magnetic Resonance Imager spectrometer incorporating Bruker AVANCE III electronics and ParaVision software version 5.1. Briefly, animals were anesthetized (1.5% isoflurane in a gas mixture of 30% oxygen and 70% nitrous oxide) and placed on a custom-manufactured (ASI Instruments) stereotaxic device with built-in temperature and cardiorespiratory monitoring engineered to fit a 25 mm Bruker mouse volume coil, as previously described (Fricke et al., 2006;Sirajuddin et al., 2012;Albanese et al., 2013). A T2-weighted two-dimensional anatomic locator scan was run followed by a volume-localized PRESS sequence with the following parameters: TE: 20 ms, TR: 2500 ms, averages: 1024, spectral width of 4 kHz, and 512,000 complex data points and 6 Hz line broadening, using a single voxel localized on the frontal cortex. All in vivo peak integrated areas were analyzed using Bruker's "TOPSPIN" software to assess relative differences in tissue chemistry, as described previously (Fricke et al., 2006;Sirajuddin et al., 2012;Albanese et al., 2013). The concentrations of metabolites were normalized to choline.
TUNEL. Slides were dried for 1 h before staining with Click-iT Plus TUNEL assay for in situ apoptosis detection (Fisher; C10618). Steps followed the manufacturer's instructions. A positive control slide was treated with DNase I.
Tamoxifen. 4-Hydroxytamoxifen (Sigma) was diluted in 100% ethanol and then diluted in peanut oil to a final concentration of 4 mg/ml. At 9-11 weeks of age, tamoxifen injections (1 mg) were administered intraperitoneally consecutively for 4 d ending 5 d before focal demyelination surgery.
Spinal cord demyelination. Focal demyelination was induced by injection of 1.0% lysolecithin (Sigma) diluted in sterile PBS into ventral funiculus. Mice were killed for analysis at 5, 10, or 20 d after surgery.
Cuprizone and special diets. All animals were fed a creatine-deficient amino acid diet (Envigo; TD.01084) unless otherwise specified. Demyelination was induced in 8-week-old male and female mice by adding 0.2% cuprizone (bis(cyclohexanone)oxaldihydrazone) into normal chow (LabDiet 5053) or creatine-deficient diet for 5 weeks. Cuprizone diet was replaced every 3 d to prevent stability concerns. All recovery diets used TD.01084 and added creatine or cyclocreatine (Sigma). Recovery diets after cuprizone were one of the following: normal chow, creatine-deficient diet, 2% creatine, or 0.1% cyclocreatine. Animals were weighed once every 2 d to ensure animals did not lose more than 10% body weight.
Electron microscopy (EM). Animals were perfused with EM fixation solution (4% PFA, 2% glutaraldehyde, 0.1 M sodium cacodylate buffer). Tissues were postfixed with 1% osmium tetroxide, and embedded in EmBed812. Ultrathin sections (70 nm) were poststained with uranyl acetate and lead citrate and examined in the Hitachi H7600 transmission electron microscope at 80 kV located at Georgetown University. Digital electron micrographs were recorded with the TIA software (FEI). Morphometric analysis was performed under blinded conditions by systematic uniform random sampling using 25 randomly selected images. ImageJ software (National Institutes of Health) was used to obtain axon diameter measurements from EM images taken at 5000Â magnification and g-ratios using the freehand selection tool (.80 axons per animal; n = 1). For g-ratios, the inner myelin sheath diameter was divided by the outer myelin sheath diameter.
Experimental design and statistical analyses. Images were collected on a Zeiss LSM 800 completed system confocal imager. Quantification of immunostaining was done by 1 or 2 blinded investigators using the ImageJ cell counter manually. For corpus callosum imaging, one medial and two lateral images of corpus callosum and cingulum were taken from 3 or 4 sections per slide (n = 3). For motor cortex imaging, a minimum of four images were analyzed from 3 animals. For spinal cord demyelinating lesions, the lesion was located by visualizing the accumulation of Hoechst-positive nuclei within the ventral white matter. A minimum of three lesion sections from 3 mice were analyzed for cell density. For cuprizone, three regions from four sections per animal were analyzed (n = 4). Density per square millimeter was calculated in Microsoft Excel as previously described by Chamberlain et al. (2017). All statistics were performed using Prism. Data are expressed as mean 6 SEM. Comparisons were analyzed by two-way ANOVA with Sidak's multiple comparison test, one-way ANOVA with Tukey's multiple comparison test, or two-tailed t test. Diagrams were generated using Mind The Graph.
Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Cerebral creatine synthesis depends on Gamt expression
Previous studies from our laboratory demonstrated that Gamt loss of function in mice impairs remyelination following experimental demyelinating injury (Chamberlain et al., 2017). However, these mice did not display obvious myelination defects or developmental abnormalities, suggesting that cerebral creatine levels may not have been disrupted during development. To determine whether cerebral creatine is detectable in the absence of Gamt expression, we performed 1 H-MRS analysis in the PFC of 8-week-old Gamt KO (Fig. 1a) and WT mice (Fig. 1b). MRS is a specialized, noninvasive imagingbased technique that enables the metabolic profiling of tissues in vivo. The level of creatine was also compared with the levels of glutamate/glutamine, myo-inositol, taurine, choline, and N-acetylaspartate in the mouse PFC (Fig. 1c). We found that the KO mice on a standard rodent diet displayed measurable but significantly lower creatine levels in the brain compared with WT (Fig. 1d), suggesting that dietary creatine can partially compensate for cerebral creatine levels when GAMT is missing. MRS analysis was also performed on KO and WT mice on a creatine-deficient diet. We found that WT mice on creatine-deficient diet displayed similar creatine levels as those on standard diet. By contrast, Gamt KO mice on creatine-deficient diet displayed undetectable creatine levels in the PFC (Fig. 1d). These data suggest that endogenously synthesized creatine can supply adequate cerebral creatine in the absence of dietary creatine, and that GAMT is the main enzyme responsible for creatine synthesis in the mouse brain. Moreover, our data also suggest that dietary creatine can supply cerebral creatine in the absence of Gamt expression in mice, providing an explanation for the lack of obvious myelination impairment in our previous study (Chamberlain et al., 2017). To examine the effect of Gamt deletion on postnatal CNS development, all following studies were performed in mice on creatine-deficient diet.
Oligodendrocytes are the major producers of creatine in the postnatal CNS Gamt expression does not begin until very late in rodent embryogenesis and is regionally limited before birth (Braissant et al., 2005). As rodent development progresses, Gamt spatiotemporal expression changes drastically (Tachikawa et al., 2004;Braissant et al., 2005). To effectively track the expression of Gamt and identify the cell populations that synthesize creatine endogenously, we developed a transgenic mouse model that expresses a GFP Heterozygous mice containing one copy of Gamt (Gamt GFP/1 ) were used to track CNS cell populations that normally synthesize creatine while mice with homozygous deletion (Gamt GFP/GFP ) were used for Gamt loss-of-function analysis. Gamt fl/fl mice without Cre recombinase were used as controls, and all animals were fed a creatine-deficient diet. IHC analysis for GFP coexpression in CNS cell types as indicators of endogenous creatine synthesis was performed on the cortical sections from postnatal (P) day P0 until P60.
control Gamt fl/fl mice at P21. The cerebellum was used to avoid any variability in tissue dissections between samples. We observed a complete loss of GAMT expression (26 kDa) in Gamt GFP/GFP mice compared with control, indicating that Gamt was effectively deleted in the Gamt GFP/GFP mouse CNS (Fig.  3b). We also observed a reduction of MOG expression in Gamt GFP/GFP mice compared with control, suggesting that GAMT loss of function affected the developmental myelination process (Fig. 3c,d). Next, immunostaining analysis was performed to examine the total number of oligodendrocyte lineage cells in the corpus callosum in Gamt GFP/GFP and control mice at P14 (Fig. 3e) and P21. We found that Gamt GFP/GFP mice exhibited significantly reduced OLIG2 1 oligodendrocyte lineage cell number compared with control at P14 (Fig. 3f). A significant reduction of CC1 1 mature oligodendrocytes in Gamt GFP/GFP mice was also observed at P14 and P21 (Fig. 3h). However, no significant changes in the number of OPCs labeled with either NG2 or NKX2.2 were detected in Gamt GFP/GFP mice compared with control at P7, P14, or P21 (Fig. 3i,j).
To determine whether the reduction in mature oligodendrocytes was driven by decreased proliferation or increased cell death, we performed IHC at P14 and P21. First, we conducted a TUNEL assay for apoptosis, and observed increased TUNEL staining in the corpus callosum in Gamt GFP/GFP compared with control at P14 and P21 (Fig. 4a,b). This finding was also confirmed using caspase-3 staining (Extended Data Fig. 4-1). We observed a slight but significant increase in TUNEL labeling at the corpus callosum, and most of the dying cells were OLIG2 1 oligodendrocyte lineage cells (Fig. 4c). Moreover, TUNEL-positive cells did not colocalize with markers of OPCs or mature oligodendrocytes in the Gamt GFP/GFP mice, but appeared colocalized with BCAS1, a marker of early myelinating oligodendrocytes (Fard et al., 2017) (Extended Data Fig. 4-1). To determine whether GAMT loss of function affected cell proliferation, we stained for Ki67, and found no significant difference in Ki67 expression in the corpus callosum between Gamt GFP/GFP and control mice at P7, P14, or P21 (Fig. 4d). Furthermore, we analyzed proliferation of OPCs (KI67 1 NG2 1 OLIG2 1 cells) at P7 and observed no difference between groups (Extended Data Fig. 4-1). These results suggest that endogenously synthesized creatine does not regulate OPC proliferation in the developing corpus callosum but may be involved in oligodendrocyte maturation.
Since myelination depends on lipogenesis, we also examined the expression of fatty acid synthase (FASN) in the corpus callosum (Dimas et al., 2019). We observed complete colocalization between FASN and mature oligodendrocyte marker, CC1, and unsurprisingly, decreased FASN-labeled cells in Gamt GFP/GFP mice compared with control, suggesting reduced oligodendrocyte lipogenesis in the corpus callosum (Extended Data Fig. 4-1). To determine whether the lack of Gamt expression in mice affects myelination, immunostaining analysis for MBP was performed. We found a significant decrease in MBP coverage in the corpus callosum at P21 (Fig. 4e,f). However, we found that the overall number of oligodendrocytes and extent of myelination were similar between Gamt GFP/GFP and control mice by P60 (Fig.  3h). These results suggest that the rate of myelination is slower in the absence of creatine but eventually catches up to controls in the adult CNS.
Gamt deletion leads to altered energetics and reduced BCK in neurons in the cortex of adult mice Creatine is known to play an important role in ATP buffering, while also regulating cellular energetics and glucose metabolism through AMP-kinase (AMPK) phosphorylation in muscles (Hardie et al., 2012). To determine whether GAMT loss of function alters AMPK signaling in the CNS, Western blot analysis of AMPK and phospho-AMPK in cortical tissues of Gamt GFP/GFP and control mice at P21 were performed (Fig. 5a). We found that total AMPK protein expression was unchanged in Gamt GFP/GFP mice compared with control (Fig. 5b). However, the level of phospho-AMPK compared with total AMPK expression was increased in Gamt GFP/GFP mice compared with control (Fig. 5c). A similar increase in AMPK phosphorylation was also observed in the cerebellum of Gamt GFP/GFP compared with control (Extended Data Fig. 5-1). These findings suggest a potential shift in brain bioenergetics in the absence of endogenously synthesized creatine.
Under increased energetic demand, AMPK is known to phosphorylate BCK, an enzyme that regulates the transfer of the high-energy phosphate in phosphocreatine to ADP for local ATP regeneration (Ramírez Ríos et al., 2014). It has previously been suggested that human BCK is expressed within inhibitory neurons and astrocytes but not in oligodendrocytes (Lowe et al., 2013). We confirmed that BCK is expressed in a subpopulation of NeuN 1 neurons and predominantly expressed in GFAP 1 astrocytic processes but does not colocalize with OLIG2 1 oligodendrocyte lineage cells ( Fig. 5d; Extended Data Fig. 5-2). To determine whether loss of GAMT affected BCK expression, immunostaining analysis of Gamt GFP/GFP and control mice at P60 was performed (Fig. 5d). We found that BCK expression was significantly reduced in the cortex of Gamt GFP/GFP mice compared with controls, and particularly in NeuN 1 neurons (Fig. 5e). However, this reduction was not associated with neuronal loss since we found no difference in the overall density of NeuN 1 neurons between Gamt GFP/GFP and control mice (Fig. 5f). To determine whether GAMT deletion affected axonal integrity in the adult CNS, analysis of nonphosphorylated neurofilament (SMI-32) was performed. We observed an increase in SMI-32 staining in the cortex of Gamt GFP/GFP mice compared with control (Fig. 5h,i), but no difference in total neurofilament staining (Fig. 5g). Additionally, Western blot analysis of cortex lysates also showed increased SMI-32 levels in Gamt GFP/GFP mice compared with control (Fig. 5j). These results suggest that GAMT deletion resulted in altered energetics and impaired axonal integrity in the adult CNS. Quantification showing of total AMPK protein (two-tailed t test; t = 2.884, df = 4, p = 0.045). c, Quantification of phosphorylated AMPK (two-tailed t test; t = 6.084, df = 4, p = 0.0037). Western blot of cerebellar lysates for AMPK and p-AMPK proteins is shown in Extended Data Figure 5-1. d, Images of BCK in neurons in the motor cortex at P60 in controls and Gamt GFP/GFP . e, Quantification of BCK 1 neuron density in the motor cortex at P60 (two-tailed t test; t = 4.912, df = 4, p = 0.008). f, Density of neurons in the motor cortex (p = 0.16, not significant). Expression of BCK in the adult corpus callosum is shown in Extended Data Figure 5-2. g, Intensity of neurofilament in the motor cortex at P60 (p = 0.2). h, Intensity of nonphosphorylated neurofilament (SMI-32) (two-tailed t test; t = 2.761, df = 4, p = 0.05). i, Images of neurofilament and nonphosphorylated neurofilament intensity in the motor cortex. j, Western blot of cortical lysates of SMI-32 protein at P60. Data are mean 6 SEM; n = 3 biological replicates. Scale bars: d, i, 50 mm. *p , 0.05. **p , 0.01.

Conditional deletion of Gamt in oligodendrocyte lineage cells leads to reduced oligodendrocytes and inefficient remyelination after demyelinating injury
We next determined whether Gamt expression in oligodendrocytes is required for remyelination. To this end, we crossed the Gamt fl/fl line with the tamoxifen-inducible PDGFRA-Cre ERT KO mouse line, and generated PDGFRA-Cre ERT ;Gamt fl/fl iKO mice to allow the conditional deletion of Gamt in oligodendrocyte lineage cells (Fig. 6a). To ensure dietary creatine did not influence oligodendrocyte lineage cell function during remyelination, all mice were fed a creatine-deficient diet. For oligodendrocyte lineage cell Gamt deletion, tamoxifen was injected intraperitoneally into mice at 10-11 weeks of age for 4 consecutive days before demyelination, and focal experimental demyelination was then performed by lysolecithin injection into the mouse spinal cord ventral white matter. This protocol for tracking remyelination in the spinal cord has been well characterized by our laboratory (Chamberlain et al., 2017). Sibling tamoxifen-injected Gamt fl/fl mice without the PDGFRA-Cre ERT allele were used as controls.
To confirm Gamt deletion, we examined the expression of GFP in the lesioned spinal cord of PDGFRA-Cre ERT ;Gamt fl/fl iKO and control mice. At 5 d post lesion (dpl), when OPCs are expected to migrate to and proliferate in lesions, we did not observe any GFP expression in or outside of the lesions of PDGFRA-Cre ERT ; Gamt fl/fl iKO or control mice (Fig. 6b), suggesting that Gamt is not expressed early in remyelination. The lack of GFP expression also suggested that no mature oligodendrocytes that survived the lesion expressed GFP. However, at ;10 dpl, when recruited OPCs are expected to have begun to differentiate into oligodendrocytes, we detected GFP expression only in CC1 1 oligodendrocytes in lesions and barely outside of lesions (Fig. 6c). Quantification of GFP-labeled oligodendrocytes showed ;79% of mature oligodendrocytes expressed GFP in lesions (Fig. 6d), suggesting that GFP 1 oligodendrocytes were derived from OPCs that have migrated into the lesion during remyelination. These results demonstrate the inducible GFP tagging approach using the PDGFRA-Cre ERT ;Gamt fl/fl line allows the identification of Gamt deleted oligodendrocyte lineage cells after demyelinating injury, and the tracking of newly regenerated oligodendrocytes in lesions.
To determine whether tamoxifen-induced Gamt deletion in PDGFRA-Cre ERT ;Gamt fl/fl iKO mice affected oligodendrocyte lineage cell progression in lesions, immunostaining analysis for oligodendrocyte lineage cell markers was performed. We detected a significant reduction of OLIG2 1 oligodendrocyte lineage cells and CC1 1 mature oligodendrocytes in lesions in the PDGFRA-Cre ERT ;Gamt fl/fl iKO compared with control at 10 dpl ( Fig. 6e-g). Moreover, MBP coverage in the lesion, which is an indicator of remyelination, was significantly reduced at 20 dpl (Fig. 6h,i). However, by 20 dpl, there was no longer a difference in total mature oligodendrocytes in the lesion (Fig. 6j). Although MBP coverage was still reduced in the PDGFRA-Cre ERT ;Gamt fl/fl iKO after 20 dpl, the increase in oligodendrocyte density to comparable levels as controls suggests that the loss of Gamt in oligodendrocyte lineage led to a transient delay in oligodendrocyte maturation following injury. These results suggest that endogenous creatine synthesis in oligodendrocytes influences the timing of oligodendrocyte maturation/remyelination but is not required for these processes.
Creatine or cyclocreatine supplemented diets increase mature oligodendrocytes and enhance remyelination after cuprizone-mediated demyelination To determine whether creatine gain of function affects remyelination, cuprizone demyelination was performed on WT mice fed with a creatine-deficient diet for 5 weeks (demyelination period; Group 1), followed by a switch to a diet without cuprizone containing either 2% creatine or 0.1% cyclocreatine, a planar creatine analog with greater brain penetrance, for 2 weeks (recovery period; Fig. 7a; Groups 2-4). Remyelination in all three recovery groups was compared with the creatinedeficient cuprizone group ( Fig. 7a; Group 1). The creatinedeficient cuprizone group was killed immediately following 5 weeks of cuprizone. The cuprizone demyelination model was used because it allowed us to monitor remyelination efficiency in the corpus callosum under long-term creatine or cyclocreatine treatment (Torkildsen et al., 2008). We found that both creatine and cyclocreatine treatment increased the Figure 7. Creatine and cyclocreatine supplemented recovery diets lead to increased mature oligodendrocytes and enhanced remyelination. a, Outline of diet for each mouse group for the cuprizone demyelination experiment. b, Images of OLIG2 1 CC1 1 oligodendrocytes in corpus callosum after 2 weeks of recovery diet (creatine-deficient, 2% creatine and 0.1% cyclocreatine). c, Density of mature oligodendrocytes (one-way ANOVA with Sidak's multiple comparisons; F (3,10) = 29.58, df = 13, p , 0.0001). d, Images of fluoromyelin in corpus callosum after various recovery diets. e, Quantification of fluoromyelin intensity in the different groups (one-way ANOVA with Dunnett's multiple comparisons; F (3,11) = 4.279, df = 14, p = 0.03). f, EM images of axons in cross sections of corpus callosum. Original magnification Â 5000. g, h, g-ratio analysis of creatine and cyclocreatine groups (one-way ANOVA with Tukey's multiple comparisons; F (2,237) = 40.28, df = 239, p , 0.0001). Data are mean 6 SEM; n = 3 or 4 biological replicates in a-d. Representative EM analysis in f-h from n = 1, 80 axons per animal. Scale bars: b, 50 mm; d, 100 mm; f, 500 nm. *p , 0.05. **p , 0.01. ***p , 0.001. ****p , 0.0001. number of OLIG2 1 CC1 1 oligodendrocytes (Fig. 7b,c), and significantly greater fluoromyelin staining in the corpus callosum compared with control after 2 weeks of recovery diet (Fig.  7d,e). Moreover, EM analysis revealed that creatine and cyclocreatine diets increased the extent of remyelinated axons compared with control, as well as lower g-ratios ( Fig. 7f-h). These results suggest that dietary creatine significantly enhanced the rate of CNS remyelination in mice.

Discussion
Creatine synthesis through Gamt supports oligodendrocyte maturation and survival during developmental myelination Clinical symptoms in CCDS suggest that the CNS is particularly vulnerable to creatine deficiency (Giusti et al., 2019). We have performed MRS analysis and showed that cerebral creatine levels are dependent on Gamt expression. Moreover, we have now generated a new transgenic mouse model that enables GFP tagging of cells normally displaying creatine synthesis and investigation of the effect of endogenous creatine loss on cellular function. The ability to perform cell-specific deletion of Gamt may aid in understanding the etiology behind the development of the intellectual disabilities, seizures, and behavior disorders observed in CCDS patients. We found that Gamt is not expressed in OPCs but is expressed in a small portion of early myelinating oligodendrocytes and highly expressed in mature oligodendrocytes, as was observed in previous studies (Tachikawa et al., 2018;Baker et al., 2021), and suggest that creatine synthesis may support the energetic demand required for developmental myelination. Our Gamt GFP/GFP KO model showed significant reductions in mature oligodendrocytes and myelin proteins at P14 and P21 compared with control, and coincided with increased cell death in early myelinating oligodendrocytes. However, the reduction of oligodendrocytes was no longer observed P60, suggesting that endogenous creatine synthesis is not required for myelination in mice but may be necessary to ensure that oligodendrocyte maturation and myelination occur in a timely manner in the postnatal brain (Hughes and Stockton, 2021). It is known that alterations in the CNS during critical windows of development can have long-lasting impacts on brain function and associated behavior (Marín, 2016). However, the lack of any overt behavioral perturbations in our mouse model may be attributed to the difference in neocortical development and white matter volume between humans and rodents.
We found that mice lacking Gamt expression displayed increased AMPK signaling, and that neurons in the adult CNS exhibited decreased BCK expression and increased SMI-32 staining in the cortex at P60, suggesting that creatine deficiency results in altered neuronal bioenergetics and decreased neuronal integrity (Yandamuri and Lane, 2016). It remains unknown whether oligodendrocyte-derived creatine regulates oligodendrocyte function cell-autonomously, or can be distributed to other cell types in the brain, thereby affecting neighboring cells non-cell-autonomously (Fünfschilling et al., 2012;Lee et al., 2012). Unfortunately, attempts to delete Gamt in oligodendrocyte lineage cells in newborn mice through tamoxifen injections have not been successful. Therefore, it remains to be determined whether the observed dysregulation in neuronal bioenergetics from Gamt deletion occurred from (1) a reduced local supply of creatine from oligodendrocytes, (2) oligodendrocyte dysfunction or loss during development, or (3) a potential role of Gamt in neural stem/precursor cell differentiation or function early in development. Importantly, previous studies of Gamt expression in CNS development using in situ hybridization suggest that Gamt may be expressed in low levels in neurons and astrocytes in addition to oligodendrocyte expression (Braissant et al., 2001). Although we did not observe any GFP expression in neurons in our study, we cannot completely rule out low levels of Gamt expression in neurons that were undetectable under standard confocal fluorescence microscopy, and therefore cannot be certain any neuronal changes observed resulted directly from the disruption of creatine synthesis in oligodendrocytes.
Endogenous creatine synthesis and dietary creatine supplementation support remyelination We found that creatine synthesis in oligodendrocytes is important for the remyelination process. Interestingly, following lysolecithin demyelination of tamoxifen-injected PDGFRA-Cre ERT ; Gamt fl/fl iKO mice, we saw no expression of the GFP reporter at 5 dpl and only in mature oligodendrocytes in the lesion at 10 dpl, suggesting that the GFP-tagged oligodendrocytes in lesions are newly regenerated oligodendrocytes involved in remyelination. The inducible GFP tagging approach may be useful for the identification and tracking of newly regenerated oligodendrocytes and could be incorporated into other animal models of demyelination or experimental injury. Since half of daily creatine level in humans comes from diet, we investigated whether dietary creatine or cyclocreatine, a lipophilic analog with greater brain penetrance, affects the efficiency of remyelination after cuprizone demyelination. We found that both creatine and cyclocreatine increased the number of oligodendrocytes and improved remyelination, but there was no significant difference between the two diets used. Increasing the cyclocreatine dose or limiting the length of recovery diet may allow us to see greater differences between the diets. Overall, this experiment suggests that creatine supplementation can improve the rate of remyelination.
In conclusion, we found that mature oligodendrocytes actively synthesize creatine during myelination, and that impaired creatine synthesis through Gamt loss results in delayed myelination during development and after injury. Moreover, we found that dietary creatine enhances CNS remyelination. The lack of endogenously synthesized creatine may affect neuronal energetics and function, but we cannot exclude the possibility that altered neuronal function occurred through GAMT loss in cells other than oligodendrocytes in the adult CNS. These results suggest oligodendrocyte dysfunction might be a contributor to the CNS pathophysiology observed under creatine deficiency conditions, such as CCDS.